| Assay Method Information | |
| | CDK4 and CDK6 Caliper Mobility Shift Assays |
| Description: | A Caliper Mobility Shift assay was used to determine the inhibitory activities of a compound against CDK4/cyclin D1 and CDK6/cyclin D1 in their respective kinase buffers (CDK4: 20 mM HEPES, pH 7.5, and 0.01% Triton X-100; CDK6: 50 mM HEPES, pH 7.5, and 0.0015% BRIJ-35). The CDK4/cyclin D1 and CDK6/cyclin D1 were obtained from PROQINASE and CARNA, respectively. A FAM-labeled peptide was obtained from GL. The compound at predetermined concentrations was pre-incubated with CDK4/cyclin D1 or CDK6/cyclin D1 in a 384-well microplate at room temperature for 10 min. The FAM-labeled peptide and ATP were subsequently added to initiate the kinase reactions. The final enzyme concentrations were 15 nM for CDK4 and 7.5 nM for CDK6. The final ATP concentrations were 672 mM for CDK4 and 230 mM for CDK6. The microplate was allowed to incubate at 28° C. The kinase reactions were stopped by adding a stop buffer (100 mM HEPES, pH 7.5, 0.012% BRIJ-35, 0.2% Coating Reagent 3 (Caliper Life Sciences), and 50 M EDTA). Conversion data were recorded and utilized to calculate the percent inhibition values for each compound and an IC50 value was then determined. |
| Affinity data for this assay | |
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